Alphaviruses and flaviviruses are small enveloped plus-sense RNA viruses with structurally and functionally-related membrane fusion proteins. These viruses include important human pathogens such as the encephalitic alphaviruses and chikungunya virus, and the flaviviruses dengue, yellow fever, and West Nile virus. Many of these viruses are classified as category A, B or C priority pathogens, yet in spite of their medica importance there are no available antiviral therapies. Although alphaviruses and flaviviruses are well-characterized structurally, relatively little is known about host factors involved in the viru lifecycle. Alphaviruses and flaviviruses infect cells by clathrin-mediated entry and a membrane fusion reaction triggered by the low pH in the endocytic pathway. New alphavirus particles bud through the host cell plasma membrane, while nascent flaviviruses bud into the endoplasmic reticulum. The long-term goal of this grant is to define the molecular mechanisms of alphavirus and flavivirus entry and exit and the role of host proteins in these processes. Our studies will focus on the highly-developed alphavirus experimental systems Semliki Forest virus and sindbis virus, and the emerging alphavirus pathogen chikungunya virus. Three integrated and complementary approaches will be used to address key features of alphavirus entry and exit: 1. We will define the roles of cellular proteins identified by our genome-wide siRNA screen for novel host factors involved in the alphavirus lifecycle. Hits were prioritized by bioinformatics analysis, confirmed with several alphaviruses, and differentially screened for key steps in virus infection. The mechanisms by which these host proteins promote or inhibit alphavirus entry, replication and exit will now be determined. 2. The small viral membrane protein 6K promotes alphavirus budding by an unknown mechanism that may involve direct effects and/or effects on cellular proteins. Our preliminary results show that the cellular membrane protein BST2/tetherin inhibits alphavirus release. We will define the mechanism of tetherin inhibition and determine if tetherin is antagonized by 6K or other viral proteins. We will determine the activity of 6K in directly modulating membrane curvature at the bud site. 3. Biochemical and imaging approaches will be used to study the cell biology of alphavirus exit, focusing in particular on the transport mechanism and assembly site of the viral nucleocapsid core, and on the properties of the final envelopment step and the mechanistic roles of host and viral proteins. Results will further our understanding the molecular mechanisms of alphavirus entry and exit, provide critical information on viral disease mechanisms and novel strategies for anti-viral therapy, and suggest fruitful new avenues for research on the flaviviruses.